American Heart Association estimates that valvular heart disease has a US prevalence of 2.5% and accounts for 20,000 deaths annually. Although replacement of the diseased valve with a xenogeneic glutaraldehyde- fixed valve (e.g., bovine pericardium (BP)) dramatically improves short term outcome, long term immune mediated attack results in calcification and ultimately valve failure (~10 yr lifespan). Deficiencies of curent valve replacements led the National Heart Lung and Blood Institute (NHLBI) cardiac surgery working group to recommend support for heart valve prosthesis biomaterial research. A tissue engineered heart valve utilizing un-fixed BP as a scaffold onto which patients' cells are grown (recellularization), has the potential to produce a potentially ideal heart valve. However, as noted by the NHLBI xenotransplantation working group, components which stimulate a recipient immune response (xenoantigens) represent the critical barrier to expanding clinical use of xenogeneic biomaterials. We propose a combinatorial approach (our novel antigen removal methodology combined with mesenchymal stem cell (MSC) mediated immunomodulation) for production of an immunologically-acceptable xenogeneic biomaterial for heart valve tissue engineering. Antigen removal aims to remove biomaterial xenoantigens, while leaving the extracellular matrix (ECM) intact and compatible with recellularization. We hypothesize that solubilization of xenoantigens is critical to facilitate their removal from the biomaterial. We therefore propose stepwise application of protein chemistry principles to solubilize and consequently removal BP xenoantigens. Aim 1: Remove water-soluble xenoantigens from intact BP while maintaining native ECM structure/function relationships and recellularization potential. Aim 1 will be conducted in two phases. Phase 1 will assess the effect of two novel factors on removal of water-soluble antigens from BP. Phase 2 will ensure that the resulting scaffold retains ECM properties compatible with heart valve tissue engineering. Aim 2: Remove lipid-soluble xenoantigens from intact BP in a stepwise manner after initial removal of water-soluble xenoantigens, while maintaining native ECM structure/function relationships and recellularization potential. Aim 2 will be conducted using the same 2 phased approach, with the exception that factors used in aim 2 are designed to remove lipid-soluble antigens. The resulting BP scaffold (with or without MSC recellularization) must be immunologically-acceptable following implantation in an immunocompetent recipient. We therefore propose: Aim 3: Assess the in vivo immune response to BP following stepwise antigen removal of water- and lipid-soluble xenoantigens. Assess effect of allogeneic MSC recellularization of BP following antigen removal (BP-AR) on in vivo immune response towards the biomaterial. Aim 3 utilizes an immunocompetent rabbit model for assessment of immune response to both BP-AR and MSC recellularized BP-AR. Completion of this proposal will result in a structurally integral, mechanically sound, immunologically- acceptable xenogeneic scaffold compatible with recellularization for heart valve tissue engineering.